Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.


Supplementary Materials and Methods
Intact protein mass spectrometry Intact protein mass spectrometry was performed at University of Colorado Boulder Mass Spectrometry Facility.Protein samples were directly injected onto a 2.1 x 5 mm Acquity UPLC BEH300 C4, 1.7 µm VanGuard Pre-Column (Waters), using a Waters Acquity classic UPLC.After sample loading, the column was washed at 0.2 mL/minute for 3 minutes with 3% (v/v) acetonitrile (ACN), then eluted with a gradient from 3% to 85% (v/v) ACN in 3 minutes then to 95% (v/v) ACN in 0.5 minutes.Detection occurred using a Synapt G2 Q-Tof mass spectrometer (Waters).Positive ES resolution mode was used for precursor mass spectra (MS1) from 200 to 2,500 m/z with: 3.0 kV capillary voltage, 80 °C source temperature, 150 °C desolvation temperature, and 550 L/h nitrogen desolvation gas flow.Deconvolution was performed using Mass Lynx v4.2 Maximum Entropy 1.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
A 15 μL sample of protein was mixed with 15 μL of 2X LDS Sample Buffer (Genscript M00676-10) supplemented with 1 mM dithiothreitol (DTT).Samples were heated at 90 °C for 2-3 min.Samples were run on a 15% polyacrylamide gel at 200 V for 35 min and stained using Coomassie blue dye with the Pierce Power Stainer system.

Cth SUMO protease expression and purification
Chemically competent C41(DE3) E. coli cells prepared with the Zymo Mix and Go Transformation Kit were transformed with pCDB302 (gifted to Addgene by Christopher Bahl, #113673).Expression cultures containing 200 mL autoinduction media (Overnight Express Instant TB Medium, Novagen 71491) supplemented with 1X trace metals (Teknova T1001) and 100 µg/mL kanamycin in 1 L baffled flasks were inoculated with 750 µL of overnight saturated starter culture and grown at 37 °C, 250 rpm for 2-3 h followed by 25 °C, 250 rpm for 24 h.Cells were harvested by centrifugation, the supernatant discarded, and the cell pellets flash frozen in liquid nitrogen.Cells were resuspended in Wash Buffer (20 mM Tris pH 8, 300 mM NaCl, 5 mM imidazole) supplemented with 0.1 mg/mL DNaseI and 1 mg/mL lysozyme then sonicated for 2 min process time with 1 s on and 1 s off at 50% amplitude (QSonica Q700).The lysate was clarified by centrifugation and the supernatant filtered through 0.45 µm syringe filters before purification via Ni-affinity chromatography.The sample was loaded on a 5 mL HisTrap column (Cytiva 17524801), washed with Wash Buffer, then eluted with Elution Buffer (20 mM Tris pH 8, 300 mM NaCl, 400 mM imidazole.The sample was dialyzed into Storage Buffer (50 mM Tris pH 8, 200 mM NaCl).After dialysis, precipitation was pelleted and the supernatant isolated.The sample was then supplemented to reach 1 mM DTT, 1 mM EDTA, and 5% glycerol and brought to 3.5 mg/mL for storage.The samples were aliquoted, flash frozen, and stored at -80 °C.Yields averaged approximately 30 mg/L autoinduction culture.To use the glycerol stock, fully thaw plate at 37 °C then mix well.Scraping frozen stocks using a multichannel pipette did not yield consistent results in successfully inoculating all wells of the overnight cultures.Use the thawed plate to inoculate an overnight starter culture plate by adding 10 µL to 200 µL LB supplemented with antibiotic.We have used a glycerol stock > 3 times; however, repeated freeze-thaw cycles will decrease cell viability.5. Detailed inoculation and expression protocol with linked OT-2 scripts.

-Inoculation and Expression
1. Prepare autoinduction media following manufacturer's instructions: a. 15 g Overnight Express™ Instant TB Medium, 3.75 mL 60% glycerol, bring to 250 mL with water in a 500 mL bottle b.Microwave until mixture is boiling for 30 s total.c.Cool to room temperature.d.Supplement to reach necessary antibiotic levels (for kanamycin in autoinduction expressions, 2X is recommended) and 1X trace metals.2. Add 2 mL of autoinduction media to each well of four 24-deep-well plates.

OT-2 -Option 1: Plate-to-plate inoculation
This OT-2 protocol uses a single pipette to transfer the inoculum from the overnight starter plate to the expression plates.This protocol requires the OT-2 p300 single channel pipette.
Link: Plate-to-plate inoculation OT-2 -Option 2: Row-swap inoculation (must be reversed later in transfer step) This OT-2 protocol uses a half-loaded multichannel pipette to transfer the inoculum from the overnight starter plate to the expression plates.This protocol requires the OT-2 p300 multichannel pipette.

OT-2 -Option 3: Cherry-picking inoculation
This OT-2 protocol uses a single channel pipette to transfer the inoculum from up to 3 different overnight starter plates to the expression plates.An Excel template is provided to specify the source and destination wells.This protocol requires the OT-2 p300 single channel pipette.
2. Discard supernatant by plate inversion -used a rapid, smooth motion to avoid spillover.
3. Add 1.5 mL of Lysis Buffer to each well (manually or OT-2 Lysis buffer addition) 4. Shake plates at 18 °C, 300 rpm (19 mm orbit) for 1 h to resuspend cells.5. Wash 8 mL of Ni-charged magnetic beads 3x with 50 mL Wash Buffer and resuspend in final volume of 8 mL. 6. Add 70 µL of the washed magnetic beads to each well.The magnetic beads settle quickly, resuspend beads before each transfer to ensure even addition.7. Shake plates at 18 °C, 250 rpm (19 mm orbit) for minimum 2 h for binding.

Remove supernatant
Option 1: Manual aspiration 1. Pull down magnetic beads using the OT-2 magnetic module or the home-built magnetic module.2. Using a large volume multichannel pipette (1.2 mL), aspirate the supernatant and discard.3. Add 300 uL Wash Buffer to each well.

OT-2 -Option 2: 1-plate aspiration
This OT-2 protocol uses a multichannel pipette to aspirate the supernatant and add Wash Buffer for one 24-well plate at a time.This protocol requires the OT-2 Magnetic Module and a p300 multichannel pipette.

OT-2 -Option 3: 4-plate aspiration
This OT-2 protocol uses a multichannel pipette to aspirate the supernatant and add Wash Buffer for four 24-well plates at a time.This protocol requires four home-built magnetic modules (Supplementary Figure 1) and a p300 multichannel pipette.
Link: 4-plate aspiration 9. Briefly shake plates to resuspend magnetic beads.7. Detailed transfer protocol from four 24-well plates to one 96-well plate with linked OT-2 scripts.

-Transfer 1. Transfer to 96-well plate
Option 1: Manually transfer 1. Manually transfer each well from four 24-well plates back to the corresponding well in one 96-well plate.

OT-2 -Option 2: Plate-to-plate transfer
This OT-2 protocol uses a single channel pipette to transfer the magnetic beads from four 24-well plates to one 96-well plate.This protocol requires a p1000 single channel pipette.
Link: Plate-to-plate transfer

OT-2 -Option 3: Row-swap transfer
This OT-2 protocol uses a half-loaded multichannel pipette to transfer the magnetic beads from four 24-well plates to one 96-well plate.This protocol requires a p300 multichannel pipette.
Link: Row-swap transfer Notes These OT-2 protocols rely on very accurate labware calibration to ensure the entire contents of the well are aspirated from the well bottom.13.Summary of intact mass spectrometry data for selected enzymes.Supplementary Figure 18.SDS-PAGE analysis of multiple stages of purification for BhrPETase, TurboPETase, PES-H1, PES-H1L92F/Q94Y, LCC, IsPETase, and TfCut2.LCC and TfCut2 were included as representative examples of a low and high yielding successfully purified protein, respectively.Purified protein (lane P for each sample) was loaded at a higher concentration to enable better visualization compared to Supplementary Figure 17.Other then TfCut2, no clear evidence of an overexpression band (labeled with a red arrow) is seen in the whole cell lysate, suggesting that these proteins were low expressing.A minor amount of purified protein (purple arrows) and SUMO tag (green arrows) is seen for all samples considered 'unsuccessfully' purified.Some samples showed evidence of incomplete cleavage (orange arrows) in the imidazole elution of the magbeads post-cleavage.

Supplementary Table
-chill a 96-deep-well plate at -20 °C.2. Prepare plasmids: a. Centrifuge to pellet lyophilized powder or liquid.b.Resuspend or dilute to achieve plasmid concentration ~10 ng/µL (this can be very flexible).3. Thaw competent cells on ice.(Prepared with Zymo Mix and Go Transformation Kit) 4. Transform Option 1: Manual Transformation 1. Transfer 50 µL of competent cells to each well.2. Add 5 µL of desired plasmid to each well.3. Incubate on ice 5-10 min.OT-2 -Option 2: Plate-to-plate transformation This OT-2 protocol uses a multichannel pipette to transfer the plasmids from the plasmid library plate to the transformation plate in a direct plate stamp.This protocol requires the OT-2 Temperature Module GEN2, p20 multichannel pipette.Link: Plate-to-plate transformation OT-2 -Option 3: Cherry-picking transformation This OT-2 protocol uses a single channel pipette to transfer plasmids from up to 3 different plasmid plates to the transformation plate.An Excel template is provided to specify the source and destination wells.This protocol requires the OT-2 Temperature Module GEN2 and the p20 single channel pipette.Link: Cherry-picking transformation 5. Add 150 µL LB without antibiotics.6. Seal with breathable seal and shake at 30 °C for 1 h, 350 rpm.* 7. Add 150 µL LB with 2X antibiotics.8. Seal with breathable seal and grow overnight for two nights at 30 °C, 350 rpm.*Notes *This step is optimized for a standard shaker (19 mm orbit).If using a plate shaker (3 mm orbit) timing may need to be adjusted.To save transformants: Save the transformation overnight plate as a glycerol stock by adding equal volume of a 60% sterile glycerol/H2O solution.Store directly at -80 °C.

Table 4 .
Detailed transformation protocol with linked OT-2 scripts.
Intact mass spectrometry for TfCut2 with the top showing the full charge ladder and the bottom showing the deconvoluted spectrum.Intact mass spectrometry for TfCut2S121P/D174S/D204P with the top showing the full charge ladder and the bottom showing the deconvoluted spectrum.